Lam Son Nguyen

Mutations in UPF3B , a member of the nonsense-mediated mRNA decay complex, cause syndromic and nonsyndromic mental retardation

Nonsense-mediated mRNA decay (NMD) is of universal biological significance. It has emerged as an important global RNA, DNA and translation regulatory pathway. By systematically sequencing 737 genes (annotated in the Vertebrate Genome Annotation database) on the human X chromosome in 250 families with X-linked mental retardation, we identified mutations in the UPF3 regulator of nonsense transcripts homolog B (yeast) (UPF3B) leading to protein truncations in three families: two with the Lujan-Fryns phenotype and one with the FG phenotype. We also identified a missense mutation in another family with nonsyndromic mental retardation. Three mutations lead to the introduction of a premature termination codon and subsequent NMD of mutant UPF3B mRNA. Protein blot analysis using lymphoblastoid cell lines from affected individuals showed an absence of the UPF3B protein in two families. The UPF3B protein is an important component of the NMD surveillance machinery. Our results directly implicate abnormalities of NMD in human disease and suggest at least partial redundancy of NMD pathways.

A UPF3-mediated regulatory switch that maintains RNA surveillance

Nonsense-mediated decay (NMD) is an RNA decay pathway that downregulates aberrant mRNAs and a subset of normal mRNAs. The regulation of NMD is poorly understood. Here we identify a regulatory mechanism acting on two related UPF (up-frameshift) factors crucial for NMD: UPF3A and UPF3B. This regulatory mechanism, which reduces the level of UPF3A in response to the presence of UPF3B, is relieved in individuals harboring UPF3B mutations, leading to strongly increased steady-state levels of UPF3A. UPF3A compensates for the loss of UPF3B by regulating several NMD target transcripts, but it can also impair NMD, as it competes with the stronger NMD activator UPF3B for binding to the essential NMD factor UPF2. This deleterious effect of UPF3A protein is prevented by its destabilization using a conserved UPF3B-dependent mechanism. Together, our results suggest that UPF3A levels are tightly regulated by a post-transcriptional switch to maintain appropriate levels of NMD substrates in cells containing different levels of UPF3B.

Contribution of copy number variants involving nonsense-mediated mRNA decay pathway genes to neuro-developmental disorders

The nonsense-mediated mRNA decay (NMD) pathway functions not only to degrade transcripts containing premature termination codons (PTC), but also to regulate the transcriptome. UPF3B and RBM8A, important components of NMD, have been implicated in various forms of intellectual disability (ID) and Thrombocytopenia with Absent Radius (TAR) syndrome, which is also associated with ID. To gauge the contribution of other NMD factors to ID, we performed a comprehensive search for copy number variants (CNVs) of 18 NMD genes among individuals with ID and/or congenital anomalies. We identified 11 cases with heterozygous deletions of the genomic region encompassing UPF2, which encodes for a direct interacting protein of UPF3B. Using RNA-Seq, we showed that the genome-wide consequence of reduced expression of UPF2 is similar to that seen in patients with UPF3B mutations. Out of the 1009 genes found deregulated in patients with UPF2 deletions by at least 2-fold, majority (95%) were deregulated similarly in patients with UPF3B mutations. This supports the major role of deletion of UPF2 in ID. Furthermore, we found that four other NMD genes, UPF3A, SMG6, EIF4A3 and RNPS1 are frequently deleted and/or duplicated in the patients. We postulate that dosage imbalances of these NMD genes are likely to be the causes or act as predisposing factors for neuro-developmental disorders. Our findings further emphasize the importance of NMD pathway(s) in learning and memory.

Whole-exome sequencing points to considerable genetic heterogeneity of cerebral palsy

Cerebral palsy (CP) is a common, clinically heterogeneous group of disorders affecting movement and posture. Its prevalence has changed little in 50 years and the causes remain largely unknown. The genetic contribution to CP causation has been predicted to be ~2%. We performed whole-exome sequencing of 183 cases with CP including both parents (98 cases) or one parent (67 cases) and 18 singleton cases (no parental DNA). We identified and validated 61 de novo protein-altering variants in 43 out of 98 (44%) case-parent trios. Initial prioritization of variants for causality was by mutation type, whether they were known or predicted to be deleterious and whether they occurred in known disease genes whose clinical spectrum overlaps CP. Further, prioritization used two multidimensional frameworks—the Residual Variation Intolerance Score and the Combined Annotation-dependent Depletion score. Ten de novo mutations in three previously identified disease genes (TUBA1A (n=2), SCN8A (n=1) and KDM5C (n=1)) and in six novel candidate CP genes (AGAP1, JHDM1D, MAST1, NAA35, RFX2 and WIPI2) were predicted to be potentially pathogenic for CP. In addition, we identified four predicted pathogenic, hemizygous variants on chromosome X in two known disease genes, L1CAM and PAK3, and in two novel candidate CP genes, CD99L2 and TENM1. In total, 14% of CP cases, by strict criteria, had a potentially disease-causing gene variant. Half were in novel genes. The genetic heterogeneity highlights the complexity of the genetic contribution to CP. Function and pathway studies are required to establish the causative role of these putative pathogenic CP genes.

Mutations in USP9X Are Associated with X-Linked Intellectual Disability and Disrupt Neuronal Cell Migration and Growth

With a wealth of disease-associated DNA variants being recently reported, the challenges of providing their functional characterization are mounting. Previously, as part of a large systematic resequencing of the X chromosome in 208 unrelated families with nonsyndromic X-linked intellectual disability, we identified three unique variants (two missense and one protein truncating) in USP9X. To assess the functional significance of these variants, we took advantage of the Usp9x knockout mouse we generated. Loss of Usp9x causes reduction in both axonal growth and neuronal cell migration. Although overexpression of wild-type human USP9X rescued these defects, all three USP9X variants failed to rescue axonal growth, caused reduced USP9X protein localization in axonal growth cones, and (in 2/3 variants) failed to rescue neuronal cell migration. Interestingly, in one of these families, the proband was subsequently identified to have a microdeletion encompassing ARID1B, a known ID gene. Given our findings it is plausible that loss of function of both genes contributes to the individuals phenotype. This case highlights the complexity of the interpretations of genetic findings from genome-wide investigations. We also performed proteomics analysis of neurons from both the wild-type and Usp9x knockout embryos and identified disruption of the cytoskeleton as the main underlying consequence of the loss of Usp9x. Detailed clinical assessment of all three families with USP9X variants identified hypotonia and behavioral and morphological defects as common features in addition to ID. Together our data support involvement of all three USP9X variants in ID in these families and provide likely cellular and molecular mechanisms involved.

ZC4H2 Mutations Are Associated with Arthrogryposis Multiplex Congenita and Intellectual Disability through Impairment of Central and Peripheral Synaptic Plasticity

Arthrogryposis multiplex congenita (AMC) is caused by heterogeneous pathologies leading to multiple antenatal joint contractures through fetal akinesia. Understanding the pathophysiology of this disorder is important for clinical care of the affected individuals and genetic counseling of the families. We thus aimed to establish the genetic basis of an AMC subtype that is associated with multiple dysmorphic features and intellectual disability (ID). We used haplotype analysis, next-generation sequencing, array comparative genomic hybridization, and chromosome breakpoint mapping to identify the pathogenic mutations in families and simplex cases. Suspected disease variants were verified by cosegregation analysis. We identified disease-causing mutations in the zinc-finger gene ZC4H2 in four families affected by X-linked AMC plus ID and one family affected by cerebral palsy. Several heterozygous females were also affected, but to a lesser degree. Furthermore, we found two ZC4H2 deletions and one rearrangement in two female and one male unrelated simplex cases, respectively. In mouse primary hippocampal neurons, transiently produced ZC4H2 localized to the postsynaptic compartment of excitatory synapses, and the altered protein influenced dendritic spine density. In zebrafish, antisense-morpholino-mediated zc4h2 knockdown caused abnormal swimming and impaired α-motoneuron development. All missense mutations identified herein failed to rescue the swimming defect of zebrafish morphants. We conclude that ZC4H2 point mutations, rearrangements, and small deletions cause a clinically variable broad-spectrum neurodevelopmental disorder of the central and peripheral nervous systems in both familial and simplex cases of both sexes. Our results highlight the importance of ZC4H2 for genetic testing of individuals presenting with ID plus muscle weakness and minor or major forms of AMC.

Profiling olfactory stem cells from living patients identifies miRNAs relevant for autism pathophysiology

BackgroundAutism spectrum disorders (ASD) are a group of neurodevelopmental disorders caused by the interaction between genetic vulnerability and environmental factors. MicroRNAs (miRNAs) are key posttranscriptional regulators involved in multiple aspects of brain development and function. Previous studies have investigated miRNAs expression in ASD using non-neural cells like lymphoblastoid cell lines (LCL) or postmortem tissues. However, the relevance of LCLs is questionable in the context of a neurodevelopmental disorder, and the impact of the cause of death and/or post-death handling of tissue likely contributes to the variations observed between studies on brain samples.MethodsmiRNA profiling using TLDA high-throughput real-time qPCR was performed on miRNAs extracted from olfactory mucosal stem cells (OMSCs) biopsied from eight patients and six controls. This tissue is considered as a closer tissue to neural stem cells that could be sampled in living patients and was never investigated for such a purpose before. Real-time PCR was used to validate a set of differentially expressed miRNAs, and bioinformatics analysis determined common pathways and gene targets. Luciferase assays and real-time PCR analysis were used to evaluate the effect of miRNAs misregulation on the expression and translation of several autism-related transcripts. Viral vector-mediated expression was used to evaluate the impact of miRNAs deregulation on neuronal or glial cells functions.ResultsWe identified a signature of four miRNAs (miR-146a, miR-221, miR-654-5p, and miR-656) commonly deregulated in ASD. This signature is conserved in primary skin fibroblasts and may allow discriminating between ASD and intellectual disability samples. Putative target genes of the differentially expressed miRNAs were enriched for pathways previously associated to ASD, and altered levels of neuronal transcripts targeted by miR-146a, miR-221, and miR-656 were observed in patients’ cells. In the mouse brain, miR-146a, and miR-221 display strong neuronal expression in regions important for high cognitive functions, and we demonstrated that reproducing abnormal miR-146a expression in mouse primary cell cultures leads to impaired neuronal dendritic arborization and increased astrocyte glutamate uptake capacities.ConclusionsWhile independent replication experiments are needed to clarify whether these four miRNAS could serve as early biomarkers of ASD, these findings may have important diagnostic implications. They also provide mechanistic connection between miRNA dysregulation and ASD pathophysiology and may open up new opportunities for therapeutic.

Rare copy number variation in cerebral palsy

Recent studies have established the role of rare copy number variants (CNVs) in several neurological disorders but the contribution of rare CNVs to cerebral palsy (CP) is not known. Fifty Caucasian families having children with CP were studied using two microarray designs. Potentially pathogenic, rare (<1% population frequency) CNVs were identified, and their frequency determined, by comparing the CNVs found in cases with 8329 adult controls with no known neurological disorders. Ten of the 50 cases (20%) had rare CNVs of potential relevance to CP; there were a total of 14 CNVs, which were observed in <0.1% (<8/8329) of the control population. Eight inherited from an unaffected mother: a 751-kb deletion including FSCB, a 1.5-Mb duplication of 7q21.13, a 534-kb duplication of 15q11.2, a 446-kb duplication including CTNND2, a 219-kb duplication including MCPH1, a 169-kb duplication of 22q13.33, a 64-kb duplication of MC2R, and a 135-bp exonic deletion of SLC06A1. Three inherited from an unaffected father: a 386-kb deletion of 12p12.2-p12.1, a 234-kb duplication of 10q26.13, and a 4-kb exonic deletion of COPS3. The inheritance was unknown for three CNVs: a 157-bp exonic deletion of ACOX1, a 693-kb duplication of 17q25.3, and a 265-kb duplication of DAAM1. This is the first systematic study of CNVs in CP, and although it did not identify de novo mutations, has shown inherited, rare CNVs involving potentially pathogenic genes and pathways requiring further investigation.

Mutations of protocadherin 19 in female epilepsy (PCDH19-FE) lead to allopregnanolone deficiency

Protocadherin 19 (PCDH19) female limited epilepsy (PCDH19-FE; also known as epilepsy and mental retardation limited to females, EFMR; MIM300088) is an infantile onset epilepsy syndrome with or without intellectual disability (ID) and autism. We investigated transcriptomes of PCDH19-FE female and control primary skin fibroblasts, which are endowed to metabolize neurosteroid hormones. We identified a set of 94 significantly dysregulated genes in PCDH19-FE females. Intriguingly, 43 of the 94 genes (45.7%) showed gender-biased expression; enrichment of such genes was highly significant (P = 2.51E-47, two-tailed Fisher exact test). We further investigated the AKR1C1-3 genes, which encode crucial steroid hormone-metabolizing enzymes whose key products include allopregnanolone and estradiol. Both mRNA and protein levels of AKR1C3 were significantly decreased in PCDH19-FE patients. In agreement with this, the blood levels of allopregnanolone were also (P < 0.01) reduced. In conclusion, we show that the deficiency of neurosteroid allopregnanolone, one of the most potent GABA receptor modulators, may contribute to PCDH19-FE. Overall our findings provide evidence for a role of neurosteroids in epilepsy, ID and autism and create realistic opportunities for targeted therapeutic interventions.

CCDC22: a novel candidate gene for syndromic X-linked intellectual disability

X-linked intellectual disability (XLID), defined as clinical ID combined with a pedigree consistent with X-linked inheritance, is a genetically heterogeneous condition that affects more than 10% of males with ID. Currently there are at least 92 genes known to cause XLID,1–3 yet a large proportion of XLID cases remain unexplained, as each of the XLID genes identified so far only accounts for a small fraction (< 1%) of affected individuals. Given that about one third of mutations affect gene expression levels,4 we reasoned that transcriptome profiling of lymphoblast cell lines from XLID patients may highlight genes harboring disease-causing mutations and may be an efficient follow-up method for rare sequence variants of unknown functional significance. We analyzed expression profiles of lymphoblast cell lines from 64 XLID patients, including 13 cases that were part of a recent X-chromosome exon re-sequencing study5 (Supplementary Methods, Supplementary Table 1). We found polyglutamine-binding protein 1 (PQBP1), a gene previously implicated in XLID,6,7 to be significantly downregulated in two cases (Supplementary Table 2), and confirmed an exon 4 (AG)2 deletion as the cause of mRNA downregulation in both instances. PQBP1 mutations cause a sydromic form of XLID commonly referred to as Renpenning syndrome.8 The specific mutation we describe here has been previously shown to cause XLID,6 and has also been proven to decrease mRNA levels by nonsense-mediated mRNA decay,7 thus being likely to be detected by assessment of gene expression. The cases for which we identified PQBP1 mutations were not part of the cohort studied by Tarpey et al. (Supplementary Table 1). We further asked whether any of the non-recurrent sequence variants identified by the exon re-sequencing study in the 13 overlapping cases was associated with a significant alteration of mRNA levels. We found that CCDC22, which encodes a coiled-coil domain protein of unknown function, was significantly downregulated (Figure 1c, Supplementary Figure 1) in one of our XLID patients. A CCDC22 non-recurrent sequence variant c.49A > G/p.T17A had been identified in a proband from the same family by the Sanger re-sequencing study, but its functional significance had not been determined.5 The proband belongs to a large family (55 individuals) with six affected males over three generations (Figure 1a). Notably, female carriers in this family show highly skewed X-chromosome inactivation (data not shown). Linkage analysis delineated a 58.4-Mb linkage interval (logarithm in base 10 of odds (LOD) score = 2.7). Whole X-chromosome exon re-sequencing identified four non-recurrent mis-sense variants within the linkage interval: CCDC22 c.49A> G/p.T17A, FAM121A (APOOL) c.40G> T/p.A14S, LOC402414 c.453T> A/p.S151R, ABCB7 c.941G> A/p.R314Q.5 Although these sequence variants were predicted to be neutral for protein function (Polyphen, data not shown), we found that the CCDC22 change was associated with a fivefold decrease in mRNA level (Figure 1c, Supplementary Figure 1 and Supplementary Table 3). No other gene in the 58.4-Mb linkage interval showed significant downregulation of mRNA levels (Supplementary Figure 2a). We sequenced CCDC22 in all available family members and found that the c.49A>G change segregates with the disease consistent with its location within the linkage interval. Figure 1 CCDC22 mutation causes X-linked intellectual disability (XLID) in a large pedigree. (a) The pedigree of the IGOLD #586 family shows six affected males over three generations. The case analyzed by expression profiling in this study (ID11) is the case from ... The c.49A >G sequence variant is located within exon 1, two base pairs away from the 5′ splice site of intron 1 of CCDC22 (Figure 1b) and is predicted to significantly decrease the splicing efficiency at the corresponding 5′ splice site (splice site score decrease from 0.9 to 0.7, using NNsplice9; http://www.fruitfly.org/seq_tools/splice.html). We found abnormally spliced transcripts retaining intron 1 to be much more abundant in cases harboring the c.49A >G mutation than in controls (Figure 1c, Supplementary Methods, Supplementary Figure 1), demonstrating that the mutation impedes efficient alternative splicing at the 5′ splice site. The abnormally spliced transcripts with intron 1 retention contain several in-frame premature terminal codons, which would likely cause the transcript to be degraded by nonsense-mediated mRNA decay surveillance (NMD). Alternatively, the splicing defect could have a negative impact on the transcription efficiency of CCDC22.10–12 Inhibition of NMD by cyclohexamide did not significantly affect the total CCDC22 mRNA level or the abundance of the abnormally spliced isoform (Supplementary Figure 1), indicating that NMD did not have a major role in CCDC22 mRNA downregulation. Recent studies have suggested that abnormal transcripts with retained intron(s), having failed the nuclear quality control mechanism, are not exported into the cytoplasm and thus are not degraded by NMD.13–17 In addition, splicing events proximal to the transcription start sites have been shown to be important for efficient recruitment of basal transcription factors.10–12 We thus suspect that the c.49A >G change near the first splice site removes the positive feedback necessary for efficient transcription of CCDC22. To test whether CCDC22 downregulation occurred frequently in the general population we analyzed CCDC22 expression level in 52 age-matched control males using genome-wide expression data from lymphoblast cell lines from an AGRE cohort (Methods). None of the controls showed significant downregulation of CCDC22 mRNA level (Supplementary Figure 2b). The phenotype of the IGOLD #586 family is consistent with syndromic XLID. In addition to intellectual disability, affected individuals have cardiac abnormalities (atrial septal defect, ventricular septal defect, dextrocardia), skeletal abnormalities (hypoplastic distal phalanges, syndactyly, hip subluxation, scoliosis) and specific facial features (Table 1). Table 1 Phenotype characterization of the IGOLD 586 family CCDC22 is a ubiquitously expressed coiled-coil domain protein (Supplementary Figure 3). Although the function of CCDC22 is currently poorly defined, CCDC22 has been shown to interact in vitro with copines, a family of calcium-dependent membrane-binding proteins, via its coiled-coil domain18 as well as the Nance–Horan syndrome protein.19 In the rat brain, CCDC22 is expressed in multiple regions including the prefrontal and somatosensory cortex, dentate gyrus and thalamus,20 and CCDC22-specific antibodies stain primarily axons.20,21 In the rat spinal cord, CCDC22 is primarily expressed in the dorsal columns, as well as in ipsilateral motor neurons after sciatic nerve trans-section,21 suggesting a role for this gene in neuronal injury response. To gain further insight into the function of CCDC22, we took a bioinformatics approach. To identify genes that are functionally related to, or potentially interact with CCDC22 in the developing human brain, we used a recently published human fetal brain transcriptome dataset22 and queried which are the nearest neighbors of CCDC22 by co-expression topological overlap.23 Figure 1d shows the genes co-expressed with CCDC22 in the human fetal brain. Remarkably, this module contains genes that have been implicated in hereditary cardiac and skeletal disorders, the main classes of extra-central nervous system pathological changes observed in family IGOLD #586. The IGOLD #586 case was the only one in the X-chromosome re-sequencing cohort of 208 with a CCDC22 mutation, suggesting that mutations of CCDC22 are a rare cause of XLID. Here we highlight CCDC22 as a novel XLID candidate gene for future targeted re-sequencing studies and propose that the mRNA downregulation associated with the described mutation likely results from reduced transcriptional efficiency rather than nonsense-mediated mRNA decay.